A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.
A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.
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DOI：
10.1093/humrep/dev300
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发表时间：
2016-02
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影响因子：
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通讯作者：
Jovanović A
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作者：
Fernandes G;Dasai N;Kozlova N;Mojadadi A;Gall M;Drew E;Barratt E;Madamidola OA;Brown SG;Milne AM;Martins da Silva SJ;Whalley KM;Barratt CL;Jovanović A
靶向ATP敏感性K+（KATP）通道的药物能否阻止体外条件下人类中期II（MII）卵母细胞中可能发生的细胞内Ca 2+的任何自发增加？吡那地尔（一种KATP通道开放剂）和格列本脲（一种KATP通道阻断剂）可防止人MII卵母细胞内Ca 2+的自发增加。卵母细胞的质量和在辅助生殖技术（ART）实验室中体外处理过程中保持这种质量对于成功的胚胎发育和健康的活产至关重要。维持Ca 2+稳态对细胞健康至关重要，细胞内Ca 2+水平升高是细胞应激的公认指标。从2013年10月至2015年7月，使用了IVF/ICSI治疗期间收集的未能受精的多余人卵母细胞（n = 102）。所有实验均在成熟（MII）卵母细胞上进行。在以下实验组中监测卵母细胞中细胞内Ca 2+水平的动态：（i）对照，（ii）二甲亚砜（DMSO;用于溶解吡那地尔、格列本脲和2，4-二硝基苯酚（DNP）），（iii）吡那地尔，（iv）格列本脲，（v）DNP：氧化磷酸化抑制剂，（vi）吡那地尔和DNP，以及（vii）格列本脲和DNP。作为常规治疗的一部分，在镇静下在辅助受孕单元从年龄为34.1 ± 0.6岁的健康女性（平均值± SD）收集卵母细胞，n = 41。临床使用的剩余部分捐赠用于研究。将卵母细胞加载Fluo-3 Ca 2+敏感染料，并通过激光共聚焦显微镜以10 min间隔监测2 h。卵母细胞采集与开始Ca 2+监测之间的时间为80.4 ± 2.1 h。在体外条件下，细胞内Ca 2+水平增加，没有故意的挑战，如Fluo-3荧光从61.0 ± 11.8 Au（Au =任意单位; n = 23）增加到监测2小时后的91.8 ± 14.0 Au（n = 19; P < 0.001）所示。吡那地尔（100 µM）可抑制Ca 2+的这种增加（实验开始时为85.3 ± 12.3 Au，实验结束时为81.7 ± 11.0 Au; n = 13; P = 0.616）。格列本脲（100 µM）也可抑制Ca 2+的增加（实验开始时为74.7 ± 10.6 Au，实验结束时为71.8 ± 10.9 Au; n = 13; P = 0.851）。DNP（100 mM）诱导细胞内Ca 2+升高，格列本脲（100 µM; n = 9）可抑制该升高，但吡那地尔（100 µM; n = 5）不抑制该升高。由于临床和伦理方面的考虑，这是不可能的监测钙在MII卵母细胞后立即检索。MII卵母细胞仅在IVF或ICSI不成功后才可用于我们的实验，平均为80.4 ± 2.1 h（n = 102个卵母细胞）。由于这里使用的MII卵母细胞是那些没有成功受精的卵母细胞，它们可能是异常的，Ca 2+稳态受损，此外，Ca 2+稳态的改变可能仅与长期孵育有关。这些结果表明，卵母细胞在体外条件下的维持与细胞内Ca 2+的增加有关，这可以通过靶向KATP通道的药物来抵消。由于Ca 2+稳态对于ART的成功结局至关重要，因此这些结果表明KATP通道开放剂和阻滞剂应作为提高ART成功率的药物进行测试。邓迪大学，MRC（MR/K 013343/1，MR/012492/1），NHS Tayside。资助苏格兰国民保健服务研究金（Sarah Martins da Silva博士）。作者声明无利益冲突。
Could drugs targeting ATP-sensitive K+ (KATP) channels prevent any spontaneous increase in intracellular Ca2+ that may occur in human metaphase II (MII) oocytes under in vitro conditions? Pinacidil, a KATP channel opener, and glibenclamide, a KATP channel blocker, prevent a spontaneous increase in intracellular Ca2+ in human MII oocytes. The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca2+ homeostasis is crucial for cell wellbeing and increased intracellular Ca2+ levels is a well-established indicator of cell stress. Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca2+ levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca2+-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca2+ monitoring was 80.4 ± 2.1 h. Intracellular levels of Ca2+ increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca2+ (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca2+ (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca2+ that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). Owing to clinical and ethical considerations, it was not possible to monitor Ca2+ in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca2+ homeostasis and, furthermore, the altered Ca2+ homeostasis might have been associated solely with the protracted incubation. These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca2+, which can be counteracted by drugs targeting KATP channels. As Ca2+ homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that KATP channel openers and blockers should be tested as drugs for improving success rates of ART. University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.